Results

- Parameters affecting cellular invasion and escape from the parasitophorous vacuole by different infective forms of Trypanosoma cruzi

Formation and escape from parasitophorous vacuole by T. cruzi infective forms. Role of cytoplasmic pH - By using the centrifugation protocol developed in our laboratory, it was possible to induce a large population of parasites to rapidly associate to the target cells. Parasites were centrifuged onto the host cells, unattached parasites aspirated and the kinetics of parasitophorous vacuole formation started by monitoring the appearance of parasites labeled with anti-LAMP-1 (Fig. 1) antibodies. During a 24 h period, it was possible to distinguish at least 3 phases in the process: an initial stage of increasing numbers of labeled parasites, probably reflecting the internalization of adhered parasites; then the number of intravacuolar parasites stabilized for a period of time and then began to decrease until no labeled parasites could be detected (Fig. 2A). We will refer to these phases as parasitophorous vacuole formation, mean time of residence and escape, respectively (see Fig. 2A). Simple kinetic parameters describing the three phases were determined for both metacyclic trypomastigotes and amastigotes, in Vero and HeLa cells (Table). For the formation and escape steps, we determined the half-life of each step (T1/2f and T1/2e, for formation and escape, respectively). Since our determinations were performed on an hourly basis, we have arbitrarily assigned this value for the mean error for each experimental point. The parameters determined for metacyclic trypomastigotes (Fig. 2B) and amastigotes (Fig. 2C) shown in the Table, indicate that there were no significant differences between the two forms with either target cell. Although no significant difference in the kinetics of formation between the two target cells or infective forms were observed, we consistently found that amastigotes tended to escape faster from the parasitophorous vacuole since after 12 h, more than 90% of the amastigotes were already free in the cytoplasm, whereas at this time 20-30% of the metacyclic forms were still labeled for anti-LAMP-1 antibodies (Fig. 2A, B). The mean time of residence within the parasitophorous vacuole was usually shorter for metacyclic forms when compared to amastigotes, regardless of the target cell used. The escape phase was comparable between the two infective forms and there were no major differences between the two target cells. When the cytoplasmic pH of Vero cells was raised with chloroquine the time of residence of metacyclic trypomastigotes increased from 4 to about 10 h, whereas no effect on the kinetics of amastigotes was observed (Fig. 2B, C and Table). Interestingly, the kinetics of vacuolar escape T1/2e by either infective form was not affected by raising cytoplasmic pH (Table). Chloroquine treatment only inhibited the invasion of metacyclic trypomastigotes (Fig. 3).

Role of host cell sialic acid on parasite invasion and vacuole escape - We used CHO mutant Lec-2 cells that are deficient in sialic acid in order to examine the role of host cell sialic acid on parasite invasion and escape from the parasitophorous vacuole. The susceptibility to invasion of Lec-2 cells by both T. cruzi infective forms was essentially the same as that of the parental CHO line (Fig. 4 A, B). We then examined the escape by the two forms by counting parasites inside the parasitophorous vacuole after 3 and 6 h post-infection and verified an efficient escape from the parasitophorous vacuoles of both forms in Lec-2 cells indicated by a significant loss of labeling with anti-LAMP1 (Fig. 4 C, D).

Transialidase and hemolysin activities of metacyclic trypomastigotes and amastigotes - The transialidase activity of isolated parasites was negligible for both forms when compared to positive controls Y strain tissue culture trypomastigotes (data not shown). When we assayed isolated parasites for hemolytic (TcTOX) activity, we could only detect hemolysis by amastigotes that after 12 h caused the rupture of 90% red blood cells. By contrast, no lytic activity could be detected in metacyclic trypomastigotes (not shown).

Interference with host calcium only affects metacyclic trypomastigote invasion - Compounds that interfere with host cell Ca2+ mobilization may interfere with parasite invasion and we have confirmed these observations by using thapsigargin and calcium ionophore A23187. Inhibition of endoplasmic reticulum calcium ATPase by thapsigargin inhibited both Vero (Fig. 5A) and HeLa (not shown) cell invasion by metacyclic trypomastigote. Less pronounced inhibitory effect was obtained with A23187 (Fig. 5A). Host cell invasion by amastigotes was not affected by either compound (Fig. 5B).